Solar radiation is both a source of light for photosynthesis and UV
that can damage plant DNA. Maintaining the integrity of
DNA is of critical
importance to all organisms. Plants use both DNA repair and various shielding
strategies to minimize DNA damage; relatively little is known about the
biochemical basis of irradiation-induced DNA damage in plants (McLennan,
AG, DNA Replication in Plants, p. 135, 1987).

UV light is generally classified into 3 subgroups: UV-A (320-390nm),
UV-B (280-320nm) and UV-C (less than 280nm). The absorption spectrum of
DNA includes wavelengths from 240 to 310nm; the level of solar UV that
reaches the surface of the earth is high in the UV-A region of the spectrum,
decreases sharply in the UV-B range, and drops to nearly zero by 290nm
(McLennan, DNA Replication in Plants, p. 135, 1987). Most studies of the
effects of UV light have used wavelengths of 254nm, in the UV-C region,
although this wavelength is extremely rare in sunlight.

UV-C and UV-B produce DNA damage primarily via the formation of cyclobutane
pyrimidine dimers (PDs). Pyrimidine dimers are 80%-90% of the UV-light
induced DNA photoproducts, with most of the rest being pyrimidine(6,4)pyrimidone
(Franklin, WA and Haseltine, WA, Mut. Res. 165:1, 1986). The epidermis
of plants absorbs 95%-99% of incoming UV light; flavonoid compounds (such
as anthocyanins) and cuticular waxes are the agents of UV absorption (McLennan,
DNA Replication in Plants, p. 135, 1987). Because anthocyanins and other
flavonoids absorb light in the UV-B range, it is commonly suggested that
these compounds shield plant DNA from damage. However, this hypothesis
has not previously been tested.

We extracted anthocyanins from the purple maize line K8 (an inbred in
the W23 background) which contains all the structural genes required to
produce anthocyanins and the genes B and
Pl that confer anthocyanin
expression on nearly every tissue of the plant. For An1 prep 5g husk tissue
was frozen in liquid nitrogen, ground to a powder, and mixed with 1% HCl
in methanol. After incubation overnight at 4 C and 3 changes of extraction
buffer the final volume was 45ml; this was filtered through Whatman #1
to remove particulate matter and stored at 4 C. For An2 prep 5g husk tissue
was finely chopped and extracted with 45ml 8:1:1 methanol:acetic acid:water.
The resulting solution was passed over a Sephadex LH20 column to remove
sugars and stored at 4 C. Quercetin (Sigma) was used from a stock solution
of 10mg/ml in methanol.

Genomic DNA was prepared from BMS cell cultures and irradiated. To test
the ability of flavonoids to shield DNA, anthocyanin extracts or quercetin
solutions were sealed between quartz plates with beeswax for irradiations.
For UV-C irradiation we used one GL-15 germicidal lamp positioned 12cm
above the sample, with an output of 30 J/m2/sec. For UV-B we used a TR302
transilluminator with filter, output rated as 8 J/m2/sec and a simulator
of solar UV-B, output rated as 1 J/m2/sec. We specifically nicked the DNA
near the pyrimidine dimers using T4 endonuclease V, which was kindly supplied
by Dr. P. C. Hanawalt, PC. DNA was treated for 15 minutes at 37 degrees
in 10mM Tris pH 8.0, 100nM NaCl, 10nM EDTA, 1mg/ml BSA, 2 microliters T4
endo V (lot 25). Alkaline loading dye was added to DNA samples to denature
them; DNA was size fractionated on 0.6% alkaline gels with running buffer
composed of 30mM NaOH, 1mM EDTA. The gels were neutralized, stained with
EtBr, photographed, and the amount of unnicked DNA quantitated by densitometry.

The anthocyanins that were isolated from purple maize husk tissue by
either of the two extraction methods absorb in the UV range. When these
anthocyanins (at a concentration approximately equal to 1/3 the concentration
in K8 plants) are interposed between DNA and a UV source they protect the
DNA from PD formation (and thus from nicking by T4 endo V) (Table 1). This
protection decreases in proportion to increase in dose. Quercetin also
protects DNA from damage.

Table 1. Percent of control band densitometer trace of BMS genomic DNA
irradiated with UV-B solar simulator (with and without anthocyanin shielding).

0 min

40 min

120 min

T4endoV:

-

+

+

+

+

+

+

+

+

+

Antho:

An1

An2

Q

An1

An2

Q

Expt. 1

79± 1

100

17

86

68

50

0

35

30

28

Expt. 2

104 ±4.3

100

23

83

83

56

11

69

43

21

Control (0 min) and two doses of UV-B were used with the same anthocyanin
shielding (An1 and An2) and quercetin shielding (Q). Columns without An1
or An2 or Q headings were unshielded. T4 endo V was omitted in the (-)
lane; two or three identical such lanes were run in each experiment to
measure loading variation; the ± indicates the variation in percent
in these two or three lanes. All data were normalized to the 0 min, plus
T4 lane.

When sections of leaf tissue from purple and green plants are irradiated
with high doses of UV-C or UV-B and the level of PDs assayed as described
above, we find that the purple tissue shows less damage than green tissue.
This demonstrates that anthocyanins also protect DNA from UV damage in
vivo.

Please Note: Notes submitted to the Maize Genetics Cooperation
Newsletter may be cited only with consent of the authors

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